Laser scanning microscope system and method of setting laser-light intensity value

ABSTRACT

A method of setting a laser-light intensity value includes: emitting laser light, the laser light being excitation light, a fluorescent-dyed biological sample being irradiated with the excitation light and emitting light; detecting fluorescence emitted by the biological sample, and outputting a signal corresponding to a brightness value; prestoring relation information, the relation information including the plurality of laser-light intensity values, and information on at least one possible correlation between a phototoxicity degree and the brightness value in relation to each of the laser-light intensity values, the phototoxicity to the biological sample resulting from the laser light; generating a fluorescence image having the brightness value based on the output signal; calculating a brightness value representative of a ROI area based on the generated fluorescence image; and referring to the relation information, and determining a laser-light intensity value satisfying tolerance of the phototoxicity based on the calculated representative brightness value.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit under 37U.S.C. §120 of U.S. patent application Ser. No. 14/516,798, titled“LASER SCANNING MICROSCOPE SYSTEM AND METHOD OF SETTING LASER-LIGHTINTENSITY VALUE,” filed Oct. 17, 2014, which claims the benefit ofJapanese Priority Patent Application JP 2013-221363 filed Oct. 24, 2013,the entire contents of which are incorporated herein by reference.

BACKGROUND

The present disclosure relates to a laser scanning microscope system anda method of setting a laser-light intensity value in view of cellphototoxicity and fluorescence fading.

In recent years, laser microscopes such as confocal microscopes andmultiphoton excitation microscopes are designed in various ways tocontrol intensity values of emission from laser light sources, in orderto reduce phototoxic damages to samples such as cells.

For example, according to the technology disclosed in Japanese PatentApplication Laid-open No. 2012-212133, sectional images of a sample isobtained in order from the surface of the sample in the depth direction.A laser-light intensity value is controlled based on the fluorescencedistribution of those sectional images. In other words, the laser-lightintensity value is controlled based on the depth from the surface of thesample. As a result, according to this document, fluorescence images maynot be affected by scattering and the like resulting from the thicknessof the sample, and fluorescence images having the same brightness may beobtained.

Moreover, when a fluorescent-dyed sample is observed, a fluorescentmaterial is excited and fading of fluorescence occurs as a result, whichis problematic. For example, the technology disclosed in Japanese PatentApplication Laid-open No. 2012-128354 deals with the fading problem.According to this technology, in order to deal with fluorescence fading,when a plurality of images are taken, the laser-light intensity value ischanged from a high intensity value to a low intensity value, and thesensitivity of a photodetector is changed from high sensitivity to lowsensitivity. A high laser-light intensity value and high sensitivity areselected before fluorescence is faded, and then an image is taken. As aresult, an image of a low brightness area of a cell, i.e., a sample, maybe taken sharply. According to this technology, a plurality of imageshaving different fluorescence intensity values are taken, and thoseimages are synthesized. As a result, a synthesized image having a largedynamic range may be obtained.

SUMMARY

According to the technology of Japanese Patent Application Laid-open No.2012-212133, a laser-light intensity value is decreased in order to takea sharp image of a section close to the surface of a sample, forexample. This technology does not directly concern phototoxicity.Moreover, the technology of Japanese Patent Application Laid-open No.2012-128354 does not deal with a fluorescence-fading phenomenonresulting from irradiation with laser light, per se.

In view of this, it is desirable to variously modify laser scanningmicroscope systems in order to take images efficiently.

In view of the above-mentioned circumstances, it is desirable to providea laser scanning microscope system and a method of setting laser-lightintensity value capable of taking images efficiently.

According to an embodiment of the present disclosure, there is provideda laser scanning microscope system, including: a laser light sourcecapable of emitting laser light, the laser light being excitation light,a fluorescent-dyed biological sample being irradiated with theexcitation light and emitting light; a photodetector configured todetect fluorescence emitted by the biological sample, and to output asignal corresponding to a brightness value; and a controller apparatusincluding storage configured to prestore first relation information, thefirst relation information including the plurality of laser-lightintensity values, and information on at least one possible correlationbetween a phototoxicity degree and the brightness value in relation toeach of the laser-light intensity values, the phototoxicity to thebiological sample resulting from the laser light, and a controllerconfigured to generate a fluorescence image having the brightness valuebased on the signal output from the photodetector, to calculate abrightness value representative of a ROI area based on the generatedfluorescence image, and to refer to the first relation information, andto determine a laser-light intensity value satisfying tolerance of thephototoxicity based on the calculated representative brightness value.

According to an embodiment of the present disclosure, the storage may beconfigured to further prestore second relation information, the secondrelation information including the plurality of laser-light intensityvalues, and information on at least one possible correlation between afading degree of the fluorescence and the brightness value in relationto each of the laser-light intensity values, the fading of thefluorescence of the biological sample resulting from the laser light,and the controller is configured to select, based on the calculatedrepresentative brightness value, one of a laser-light intensity valuesatisfying a tolerance of the phototoxicity determined with reference tothe first relation information and a laser-light intensity valuesatisfying a tolerance of the fading determined with reference to thesecond relation information, the selected laser-light intensity valuebeing smaller than the other laser-light intensity value.

According to an embodiment of the present disclosure, the storage may beconfigured to prestore third relation information, the third relationinformation showing a relation between a depth and an attenuation degreeof the excitation light, the depth being between a surface of thebiological sample and a position irradiated with the excitation light,and the controller is configured to refer to the third relationinformation, and to correct the selected laser-light intensity value.

According to an embodiment of the present disclosure, the controller maybe configured to select sensitivity of the photodetector based on thecorrected laser-light intensity value.

According to an embodiment of the present disclosure, the storage may beconfigured to prestore fourth relation information, the fourth relationinformation showing relation between an elapsed time of observation ofthe biological sample and a laser-light intensity value satisfying atolerance of phototoxicity, the phototoxicity to the biological sampleresulting from the laser light, and the controller is configured toselect one of the corrected laser-light intensity value and alaser-light intensity value determined based on the elapsed time ofobservation with reference to the fourth relation information, theselected laser-light intensity value being lower than the otherlaser-light intensity value.

According to an embodiment of the present disclosure, the storage may beconfigured to prestore fifth relation information, the fifth relationinformation showing relation between an elapsed time of observation ofthe biological sample and a laser-light intensity value satisfying atolerance of fading, the fading being given to the biological sample bythe laser light, and the controller is configured to select one of thecorrected laser-light intensity value and a laser-light intensity valuedetermined based on the elapsed time of observation with reference tothe fifth relation information, the selected laser-light intensity valuebeing lower than the other laser-light intensity value.

According to an embodiment of the present disclosure, there is provideda method of setting a laser-light intensity value, including: emittinglaser light, the laser light being excitation light, a fluorescent-dyedbiological sample being irradiated with the excitation light andemitting light; detecting fluorescence emitted by the biological sample,and outputting a signal corresponding to a brightness value; prestoringrelation information, the relation information including the pluralityof laser-light intensity values, and information on at least onepossible correlation between a phototoxicity degree and the brightnessvalue in relation to each of the laser-light intensity values, thephototoxicity to the biological sample resulting from the laser light;generating a fluorescence image having the brightness value based on theoutput signal; calculating a brightness value representative of a ROIarea based on the generated fluorescence image; and referring to therelation information, and determining a laser-light intensity valuesatisfying tolerance of the phototoxicity based on the calculatedrepresentative brightness value.

As described above, according to the present technology, it is possibleto take an image efficiently in view of cell phototoxicity andfluorescence fading resulting from irradiation with laser light.

Note that in addition to the above-mentioned effect, any effectdescribed in the present disclosure may be obtained.

These and other objects, features and advantages of the presentdisclosure will become more apparent in light of the following detaileddescription of best mode embodiments thereof, as illustrated in theaccompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram schematically showing the configuration of themicroscope system 1;

FIG. 2 shows specific examples of setting information (image-takingsetting information) used to take an image, and purposes of theimage-taking setting information;

FIG. 3 shows specific examples of information (user-input information)on a sample input by a user, who performs fluorescence observation, andpurposes of the user-input information;

FIG. 4 is a flowchart illustrating the flow of entire processingperformed by the controller 51 of the system controller PC 50;

FIG. 5 is a table showing appropriate laser-light intensity valuesdepending on combinations of wavelengths of laser light and the kinds offluorescent reagents;

FIG. 6 is a flowchart illustrating the method of calculating alaser-light intensity value;

FIG. 7 is a diagram illustrating the concept of control of cell deathresulting from phototoxicity, in which the laser-light intensity valueis controlled;

FIG. 8 is a flowchart illustrating the entire processing flow of thecontroller 51 of the system controller PC 50;

FIG. 9 is a diagram schematically showing the configuration of themicroscope system 1 a;

FIG. 10 is a flowchart illustrating the entire processing flow of thecontroller 51 of the system controller PC 50; and

FIG. 11 is a flowchart illustrating the method of calculating alaser-light intensity value.

DETAILED DESCRIPTION OF EMBODIMENTS

Hereinafter, embodiments of the present disclosure will be describedwith reference to the drawings. Note that hereinafter a confocalmicroscope, i.e., one kind of fluorescence microscopes, will bedescribed as an example. Note that it does not mean that the presenttechnology is only applicable to confocal microscopes. As a matter ofcourse, the present technology is applicable to other microscopes, forexample, multiphoton microscopes or the like.

First Embodiment

[Configuration of Microscope System]

Firstly, a microscope system 1 of the present technology will bedescribed schematically. Note that the microscope system 1 is a laserscanning microscope system. FIG. 1 is a diagram schematically showingthe configuration of the microscope system 1.

The microscope system 1 includes a laser light source unit (laser lightsource) 10, a scanner unit 20, a microscope 30, a microscope controller40, and a system controller PC (Personal Computer) (controller) 50. Notethat the system controller PC 50 analyzes images and performs otherprocessing. Alternatively, an image analytical server 100 in a localnetwork or an image analytical cloud server 200 in the Internet cloudmay analyze images and perform other processing.

The laser light source unit 10 generates excitation light such that asample (biological sample) such as a fluorescently-labeled cell maygenerate fluorescence. The generated excitation light enters the scannerunit 20.

The laser light source unit 10 includes a laser controller 11. The lasercontroller 11 controls intensity values of the excitation light,light-emitting intervals, and the like. In this embodiment, the systemcontroller PC 50 determines laser-light intensity values. The systemcontroller PC 50 notifies the laser controller 11 of the determinedlaser-light intensity value via a scanner controller (described later).

The scanner unit 20 includes a scanner controller 21, a galvano mirror22, a dichroic mirror 23, and a photodetector 24.

The galvano mirror 22 changes directions of laser light such that thelaser-light scanning is performed in the X direction and the Ydirection. As a result, the excitation laser light introduced from thelaser light source unit 10 moves in the horizontal direction (XYdirections) of a sample, which is mounted on the stage 35 of themicroscope 30, and the sample is irradiated with the excitation laserlight. The laser light, whose direction is controlled by the galvanomirror 22, passes through the dichroic mirror 23. Then the laser lightenters the microscope 30. The sample is irradiated with the laser lightentered in the microscope 30. When the sample is irradiated with thelaser light, the sample is excited and generates fluorescence. Thefluorescence returns from the microscope 30 to the scanner unit 20.

The laser light and the fluorescence return from the microscope 30 tothe dichroic mirror 23. The dichroic mirror 23 reflects only thefluorescence out of them to the photodetector 24.

Generally, the photodetector 24 is a PMT (photomultiplier tube). Asdescribed above, when the sample is irradiated with the laser light, thesample is excited and generates fluorescence. The photodetector 24detects the fluorescence. Note that in a confocal microscope, a pinholeis provided on the light path in front of the photodetector 24. There isa conjugate relation between the position of the pinhole and the focalposition of the objective lens 32 (described later).

The scanner controller 21 controls the galvano mirror 22 and controlsthe photodetector 24 to scan the sample in the XY directions. Thescanner controller 21 converts the signals, which are detected by thephotodetector 24, to brightness values of the respective points on thescanned XY plane. The scanner controller 21 transmits the brightnessvalues to the system controller PC 50.

The microscope 30 includes a filter 31, the objective lens 32, the stage35, and a stage driver 34. Note that a sample is mounted on the stage35, and is observed.

The filter 31 guides the laser light entered from the scanner unit 20 tothe objective lens 32. Moreover, the sample is irradiated with thelaser, and generates fluorescence. The filter 31 guides the fluorescenceto the scanner unit 20.

The objective lens 32 collects the laser light, which enters from thescanner unit 20 via the filter 31, on the focal position of theobjective lens 32. Moreover, the objective lens 32 guides thefluorescence, which is generated by the sample, to the scanner unit 20via the filter 31.

A sample is mounted on the stage 35. The stage driver 34 moves the stage35 in the XY directions and in the Z direction. The XY directions areorthogonal to the optical axis of the objective lens 32. The Z directionis along the optical axis of the objective lens 32.

The system controller PC 50 inputs instructions on autofocus,cell-tracking exposure, and the like in the microscope controller 40.The microscope controller 40 outputs instructions for moving the stage35 to the stage driver 34 based on the instructions from the systemcontroller PC 50.

The system controller PC 50 includes a controller 51 and storage 52.

The controller 51 controls the entire microscope system 1. Thecontroller 51 synthesizes a fluorescence image based on the brightnessvalues detected by the photodetector 24 and based on the coordinatevalues on the XY plane of the detected brightness values. The controller51 analyzes the synthesized fluorescence image, calculates the optimumlaser-light intensity value, and controls the laser-light intensityvalue. Moreover, as described above, the controller 51 controls themicroscope controller 40, and causes the autofocus function to performthe cell-tracking exposure function, and the other functions of themicroscope 30. Note that a CPU (Central Processing Unit) executesprograms stored in the storage 52 to implement the controller 51.

The storage 52 includes a hard disk drive and/or a semiconductor memory.The storage 52 stores the above-mentioned programs executed by the CPU,and the fluorescence image obtained by the scanner unit 20. The storage52 further stores brightness/phototoxicity tables 52 a (first relationinformation, relation information), brightness/fading tables 52 b(second relation information), depth/laser light deterioration tables 52c (third relation information), and the like. The three kinds of tablesstored in the storage 52, i.e., the brightness/phototoxicity tables 52a, the brightness/fading tables 52 b, and the depth/laser lightdeterioration tables 52 c, will be described later.

The configuration of the microscope system 1 has been describedschematically.

[Three Kinds of Tables]

Here, the above-mentioned three kinds of tables will be described. Thosetables relate to the basic mechanism of the present technology.

According to the basic mechanism of the present technology, firstly, afluorescence image is taken. The current phototoxicity and the currentfading degree are determined based on the brightness value of a ROI(Region Of Interest) area in the taken fluorescence image. Thelaser-light intensity value is controlled such that the determinedphototoxicity and fading degree may not affect observation. Then anotherimage is taken with the controlled laser light.

Note that, for example, a taken and synthesized fluorescence image hasthirty-two blocks in a matrix. The above-mentioned ROI area is a block,which has many large brightness values (large brightness density) out ofthe thirty-two blocks. In the present technology, a laser-lightintensity value is controlled based on the brightness valuerepresentative of the ROI area. The brightness value representative ofthe ROI area may be a dynamic range value of the ROI area.

It is necessary to previously measure relations between fluorescenceintensity values (brightness values) and phototoxicitydegrees/fluorescence fading degrees obtained by using various kinds oflaser light having various intensity values. Those relations aremeasured in relation to combinations of cells (samples) to be observedand fluorescent reagents to be used for observation. After that, asdescribed above, the current phototoxicity and the current fading degreeare determined based on a brightness value. As described above, piecesof data on various combinations of cells and fluorescent reagents arecollected. This is because the fluorescence intensity values,phototoxicity degrees, and fading degrees to be obtained are differentdepending combinations.

The above-mentioned brightness/phototoxicity table 52 a shows therelation between brightness values and phototoxicity measured for eachlaser-light intensity value, as described above.

Moreover, similarly, the above-mentioned brightness/fading table 52 bshows the relation between brightness values and fading measured foreach laser-light intensity value. The brightness/fading table 52 b isalso generated for each kind of fluorescent reagents.

Note that attenuation of laser light depending on depth is measured. Thedepth is between a surface of a sample, which is irradiated with laserlight, and an observed point. The depth/laser light deterioration table52 c shows data on the attenuation of laser light relative to the depth.

Those tables are generated in relation to laser-light intensity values,the kinds of fluorescent reagents, and the kinds of samples. So a hugenumber of tables are generated.

A phototoxicity degree, a fading degree, and a laser light deteriorationdegree are obtained based on a brightness value with reference to thethree kinds of tables. Hereinafter, the phototoxicity degree, the fadingdegree, and the laser light deterioration degree will be referred to asa phototoxicity parameter, a fading parameter, and a laser-lightdeterioration parameter, respectively.

Note that, in the above description, the tables show the relationbetween brightness values and phototoxicity, the relation betweenbrightness values and fading, and the relation between brightness valuesand depth. Alternatively, if possible, functions showing those relationsmay be prepared. In the case where functions showing those relations areprepared, the storage 52 stores function-formulae instead of theabove-mentioned tables.

The three kinds of tables have been described.

[Processing Flow]

In this embodiment, the processing flow roughly includes the initialsetting process and the main image-taking process. The initial settingprocess is performed only once before the main image-taking process. Themain image-taking process is performed again and again while brightnessvalues are fed back and the laser-light intensity value is controlled.

In the following description of the processing, firstly, informationcollected in the initial setting process will be described. After that,the entire processing flow will be described.

(Information Collected in Initial Setting Process)

The controller 51 collects information during the initial settingprocess. Here, the information collected by the controller 51 will bedescribed in detail. The collected information is used in the mainimage-taking process. For example, the collected information is used toselect a laser-light intensity value to take an image for the firsttime.

FIG. 2 shows specific examples of setting information (image-takingsetting information) used to take an image, and purposes of theimage-taking setting information. Moreover, FIG. 3 shows specificexamples of information (user-input information) on a sample input by auser, who performs fluorescence observation, and purposes of theuser-input information.

Firstly, laser-wavelength information is one piece of image-takingsetting information. For example, the above-mentioned three kinds oftables may be generated in relation to laser-light intensity values and,in addition, laser wavelengths. In this case, the laser-wavelengthinformation is used as a reference to select one of the tables inrelation to laser wavelengths. Note that the tables in relation to laserwavelengths are used to sort attenuation levels of laser light.

Next, image-taking time information is another piece of image-takingsetting information. The image-taking time information is used topredict a maximum time period in which fluorescence observation isperformed.

Next, microscope-type information is another piece of image-takingsetting information. The microscope-type information distinguishesbetween a confocal microscope and a multiphoton microscope, which takesa fluorescence image. Such distinction is used to set a laser-lightintensity value because the phototoxicity of the confocal microscope islarger than the phototoxicity of the multiphoton microscope.

Next, position-in-Z-direction information is another piece ofimage-taking setting information. The position-in-Z-directioninformation is used to control a laser-light intensity value based onthe depth between a sample surface and observed position.

Next, photodetector sensitivity information is another piece ofimage-taking setting information. The photodetector sensitivityinformation is used to appropriately control a dynamic range when takingan image.

Note that the above-mentioned pieces of image-taking setting informationare necessary when taking an image except for the image-taking timeinformation. So those pieces of image-taking setting information may beobtained without fail before the main image-taking process.

Next, description will be made with reference to FIG. 3. Firstly,observed-object information is one piece of user-input information ofFIG. 3. The observed-object information is used to select some of theabove-mentioned tables. The reason is as follows. The laser-lightintensity value to be selected is different depending on the kind of asample, i.e., a general cell or a special nerve cell, for example. Notethat one brightness/phototoxicity table 52 a and one depth/laser lightdeterioration table 52 c are selected.

Next, fluorescent-dye information is another piece of user-inputinformation. Examples of fluorescent dye (fluorescent reagent) include,for example, DAPI (4′,6-diamidino-2-phenylindole), CFP (Cyan FluorescentProtein), GFP (Green Fluorescent Protein), and the like. Thefluorescent-dye information is used to select one appropriatebrightness/fading table 52 b.

Next, purpose-of-measurement information is another piece of user-inputinformation. The purpose-of-measurement information shows purpose ofobservation. Examples of the purpose of observation include measurementof a fluorescence lifetime, measurement of FRET (Fluorescence resonanceenergy transfer), calcium imaging, and the like. Thepurpose-of-measurement information is used to determine if a laser-lightintensity value is to be controlled or not in the main image-takingprocess. For example, in the case of measuring a fluorescence lifetime,a laser-light intensity value should not be controlled but should beconstant.

The image-taking setting information and the user-input information,which are collected in the initial setting process, have been describedin detail.

(Flow of Entire Processing)

Here, the flow of entire processing performed by the controller 51 ofthe system controller PC 50 will be described. FIG. 4 is a flowchartillustrating the flow of entire processing performed by the controller51 of the system controller PC 50.

Firstly, the controller 51 obtains user-input information (Step S10).The user-input information has been described in detail.

Next, the controller 51 obtains image-taking setting information (StepS20). The image-taking setting information has been described in detail.

Next, the controller 51 determines a primary laser-light intensity valueto take a fluorescence image for the first time based on the obtaineduser-input information and the obtained image-taking settinginformation. Then the determined primary laser-light intensity value isset for the laser controller 11 (Step S30).

The initial setting process includes the process of Step S10 to StepS30. The main image-taking process includes the process of Step S40 andthereafter.

Next, the controller 51 inputs instructions for taking a fluorescenceimage of a sample in the laser controller 11, the scanner controller 21,and the microscope controller 40. The controller 51 obtains a takenfluorescence image (Step S40).

Next, the controller 51 determines if image-taking is to be finished ornot (Step S50).

If image-taking is not to be finished (Step S50, N), next, thecontroller 51 analyzes the obtained fluorescence image. The controller51 selects an ROI area, and calculates a brightness value representativeof the ROI area (Step S60).

Next, the controller 51 calculates a phototoxicity parameter(laser-light intensity value) based on the calculated brightness valueand based on a brightness/phototoxicity table 52 a stored in the storage52 (Step S70).

Next, the controller 51 calculates a fading parameter (laser-lightintensity value) based on the calculated brightness value and based on abrightness/fading table 52 b stored in the storage 52 (Step S80).

The depth between the sample surface and the measured point iscalculated based on the obtained position-in-Z-direction information byusing a generally-known method. Next, the controller 51 calculates alaser-light deterioration parameter based on the depth and based on adepth/laser light deterioration table 52 c stored in the storage 52(Step S90).

Next, the controller 51 calculates a laser-light intensity value, whichis to be used when taking a next image, based on the phototoxicityparameter, the fading parameter, and the laser-light deteriorationparameter (Step S100). Note that the method of calculating a laser-lightintensity value based on the phototoxicity parameter, the fadingparameter, and the laser-light deterioration parameter will be describedlater in detail.

Next, the controller 51 resets a laser-light intensity value for takingan image with the calculated laser-light intensity value for the lasercontroller 11 (Step S110). Because the laser-light intensity value iscontrolled here, it is possible to prevent the saturation of afluorescence image from being increased and to use a dynamic rangeeffectively.

Next, the controller 51 predicts a fluorescence intensity value suchthat the optimum dynamic range may be obtained in combination with thereset laser-light intensity value. The controller 51 also resets thesensitivity of the photodetector 24 (Step S120).

After the process of Step S120, the controller 51 returns to Step S40,and takes an image of a sample based on the reset laser-light intensityvalue and the reset sensitivity.

Note that in the above-mentioned Step S30, a primary laser-lightintensity value, which is used to take a fluorescence image for thefirst time, is determined based on the obtained user-input informationand the obtained image-taking setting information. At this time, asshown in FIG. 5, there may be prepared a table showing appropriatelaser-light intensity values depending on combinations of wavelengths oflaser light and the kinds of fluorescent reagents. A laser-lightintensity value in the table, which satisfies a condition, may be aprimary laser-light intensity value.

The entire processing flow of the controller 51 of the system controllerPC 50 has been described.

(Method of Calculating Laser-Light Intensity Value)

Here, the method of calculating a laser-light intensity value of theabove-mentioned Step S100 will be described. FIG. 6 is a flowchartillustrating the method of calculating a laser-light intensity value.

Note that, in the method described below, one of the cell phototoxicityparameter and the fluorescence fading parameter having a largerinfluence is selected. A laser-light intensity value is calculated basedon the selected parameter. Here, the parameter having a larger influenceis selected as follows. For example, the parameter exhibiting thesmaller laser-light intensity value is selected.

Note that the influence of the cell phototoxicity parameter is comparedwith the influence of the fluorescence fading parameter as follows. Inthe following, the laser-light intensity value (laser-light intensityvalue satisfying tolerance of phototoxicity) required to reduce thephototoxicity is compared with the laser-light intensity value(laser-light intensity value satisfying tolerance of fading) required toreduce fading.

Then, a parameter requiring a smaller laser-light intensity value isselected. Then, a final laser-light intensity value used for resettingis calculated based on the selected parameter and a laser-lightdeterioration parameter.

Such a calculation method is used because it is necessary to determine alaser-light intensity value to reduce influence of phototoxicity, toreduce influence of fading, and to make an observation time longer. Thecriterion for calculating a laser-light intensity value to be reset ischanged depending on the cell phototoxicity parameter or thefluorescence fading parameter, which has a larger impact on a sample tobe observed.

Firstly, the controller 51 obtains a first laser-light intensity valuebased on the phototoxicity parameter calculated in Step S70 (Step S101).

Next, the controller 51 obtains a second laser-light intensity valuebased on the fading parameter calculated in Step S80 (Step S102).

Next, the controller 51 determines if the first laser-light intensityvalue is larger than the second laser-light intensity value or not (StepS103).

The controller 51 gives priority to the smaller laser-light intensityvalue, and calculates a final laser-light intensity value based on thesmaller laser-light intensity value. So if the first laser-lightintensity value is larger than the second laser-light intensity value(Step S103, Y), the controller 51 selects the fading parameter. Then,the controller 51 calculates a final laser-light intensity value usedfor resetting based on the selected fading parameter and the laser-lightdeterioration parameter (Step S104).

The final laser-light intensity value is obtained based on the followingformula, where 1roi is a brightness value representative of the ROIarea, Sf( ) is a fading parameter, z is the depth between a samplesurface and an observed position, and D( ) is a laser-lightdeterioration parameter.Final laser-light intensity value=Sf(1roi)×D(z)   (1)

The controller 51 gives priority to the smaller laser-light intensityvalue, and calculates a final laser-light intensity value based on thesmaller laser-light intensity value. To the contrary, if the firstlaser-light intensity value is not larger than the second laser-lightintensity value (Step S103, N), the controller 51 selects thephototoxicity parameter. Then, the controller 51 calculates a finallaser-light intensity value used for resetting based on the selectedphototoxicity parameter and the laser-light deterioration parameter(Step S105).

The final laser-light intensity value is obtained based on the followingformula, where 1roi is a brightness value representative of the ROIarea, Sp( ) is a phototoxicity parameter, z is the depth between asample surface and an observed position, and D( ) is a laser-lightdeterioration parameter.Final laser-light intensity value=Sp(1roi)×D(z)  (2)

Note that a final laser-light intensity value used for resetting may becalculated as follows. The phototoxicity parameter or the fadingparameter is multiplied by the laser-light deterioration parameter. Thephototoxicity parameter or the fading parameter multiplied by thelaser-light deterioration parameter corresponds to the phototoxicity orthe fading status biased in the depth direction.

The method of calculating a laser-light intensity value in theabove-mentioned Step S100 has been described.

[Concept of Laser-Light Intensity Value Control]

Here, the concept of control of cell death in relation to phototoxicityby controlling the above-mentioned laser-light intensity value will bedescribed. FIG. 7 is a diagram illustrating the concept of control ofcell death resulting from phototoxicity, in which the laser-lightintensity value is controlled.

In the graph of FIG. 7, the horizontal axis shows observation time(second). Moreover, the vertical axis shows relative laser-lightintensity values, where the normal laser-light intensity value is 1. Thevertical axis further shows the relative rate of viable cells, where thenumber of viable cells at the start of observation is 1.

Firstly, the laser-light intensity value of the normal power, i.e., 1,is maintained (straight line of “normal power” in graph), and cells areobserved. In this case, the line “cell death” shows how the rate ofviable cells changes as time passes.

The line “cell death” shows the following fact. When the laser-lightintensity value is constant, the rate of viable cells falls below 0.5after sixty seconds, for example. Then, the viable cell rate isdecreased after that.

To the contrary, the laser-light intensity value is controlled as timepasses, and the line “control” of the graph shows the laser-lightintensity values. In this case, the “controlled value” of the graphshows that cell death is prevented from occurring, and the viable cellrate is not decreased.

Note that fluorescence fading is controlled similar to the graph ofphototoxicity.

The concept of control of cell death in relation to phototoxicity bycontrolling the laser-light intensity value has been described.

[Outline of this Embodiment]

The outline of this embodiment is as follows. In other words, accordingto this embodiment, the laser scanning microscope system 1 includes: thelaser light source unit 10 capable of emitting laser light, the laserlight being excitation light, a fluorescent-dyed biological sample beingirradiated with the excitation light and emitting light; thephotodetector 24 configured to detect fluorescence emitted by thebiological sample, and to output a signal corresponding to a brightnessvalue; and the system controller PC 50 including the storage 52configured to prestore first relation information, the first relationinformation including the plurality of laser-light intensity values, andinformation on at least one possible correlation between a phototoxicitydegree and the brightness value in relation to each of the laser-lightintensity values, the phototoxicity to the biological sample resultingfrom the laser light, and the controller 51 configured to generate afluorescence image having the brightness value based on the signaloutput from the photodetector 24, to calculate a brightness valuerepresentative of a ROI area based on the generated fluorescence image,and to refer to the first relation information, and to determine alaser-light intensity value satisfying tolerance of the phototoxicitybased on the calculated representative brightness value.

Second Embodiment

Next, the second embodiment will be described. In the first embodiment,image-taking setting information and user-input information are obtainedto set a primary laser-light intensity value. To the contrary, in thisembodiment, a primary laser-light intensity value is selected withoutobtaining user-input information. Because user-input information is notobtained, the process may be performed more automatically.

Note that in this embodiment, instead of obtaining user-inputinformation, a low laser-light intensity value is set, and an image of asample is taken preliminarily. Then, the preliminarily-takenfluorescence image is recognized. As a result, information correspondingto the user-input information is obtained.

[Configuration of Microscope System]

The configuration of the microscope system of the second embodiment issimilar to the configuration of the microscope system 1 of the firstembodiment. The configuration of the microscope system of the secondembodiment will thus not be described. The configuration of themicroscope system of the second embodiment is different from that of thefirst embodiment in that the controller 51 of the system controller PC50 preliminarily takes an image of a sample and recognizes thepreliminarily-taken fluorescence image.

Note that similar to the first embodiment, not the controller 51 but theimage analytical server 100 in the local network or the image analyticalcloud server 200 in the Internet cloud may recognize the image.

The configuration of the microscope system 1 has been described above.

[Processing Flow]

The processing flow of this embodiment roughly includes the preliminaryimage-taking process and the main image-taking process. The preliminaryimage-taking process is performed only once before the main image-takingprocess. The main image-taking process is similar to that of the firstembodiment. The main image-taking process is performed again and againwhile brightness values are fed back and the laser-light intensity valueis controlled.

In the following description of the processing, firstly, informationcollected in the preliminary image-taking process will be described.After that, the part of the entire processing flow different from thefirst embodiment will be described.

(Information Collected in Preliminary Image-Taking Process)

The controller 51 collects information during the preliminaryimage-taking process. Here, the information collected by the controller51 will be described in detail. The controller 51 collects theimage-taking setting information same as that of the first embodiment,laser-light intensity value information at the time of preliminaryimage-taking, and a preliminarily-taken fluorescence image.

The image-taking setting information, the laser-light intensity valueinformation at the time of preliminary image-taking, and thepreliminarily-taken fluorescence image are used to recognize an imageafter the image is taken preliminarily, and to select a laser-lightintensity value to take an image for the first time in the mainimage-taking process, for example.

(Processing Flow (Different Part))

Here, part of the entire processing flow of the controller 51 of thesystem controller PC 50, which is different from that of the firstembodiment, will be described. FIG. 8 is a flowchart illustrating theentire processing flow of the controller 51 of the system controller PC50.

First, the controller 51 obtains image-taking setting information (StepS20). The image-taking setting information has been described in detail.

Next, the controller 51 selects a low laser-light intensity value forpreliminary image-taking (Step S21). The laser-light intensity value maybe such a value that information corresponding to the user-inputinformation may be obtained by recognizing a taken fluorescence image ofa sample.

Next, the controller 51 inputs instructions for taking a fluorescenceimage of a sample in the laser controller 11, the scanner controller 21,and the microscope controller 40. The controller 51 obtains a takenfluorescence image (Step S22).

Next, the controller 51 recognizes the fluorescence image preliminarilytaken in Step S22 (Step S23). The image is recognized based on a knowngeneral method.

For example, a plurality of samples to be observed are dyed with variousfluorescent reagents to be used for observation, and many fluorescenceimages are obtained previously. In the image recognition process, thepreliminarily-taken image is compared with those previously obtainedimages. Information corresponding to the user-input information ispresumed based on the result. In other words, the kind of the sample ofthe taken fluorescence image, the kind of the used fluorescent reagent,and the purpose of observation are presumed.

Next, the controller 51 determines the primary laser-light intensityvalue for the main image-taking process based on the obtainedimage-taking setting information and based on the presumed user-inputinformation. Then the determined primary laser-light intensity value isset for the laser controller 11 (Step S30).

The preliminarily image-taking process is different from the process ofthe first embodiment and has been described above. The process afterthat is the same as the process of the first embodiment, and descriptionthereof will be omitted.

The part of the entire processing flow of the controller 51 of thesystem controller PC 50, which is different from that of the firstembodiment, has been described.

Third Embodiment

In the above-mentioned embodiments, a brightness value of a takenfluorescence image is fed back. A phototoxicity status of a sample and afluorescence fading status are obtained based on the brightness value.Then a laser-light intensity value, which is to be used to take a nextimage, is controlled. To the contrary, in this embodiment, an elapsedtime of observation is also fed back in addition to the brightnessvalue. As a result, accuracy of controlling laser light may be improved.

Note that the graph (FIG. 7) of the first embodiment is used to feedback an elapsed time and to reflect the elapsed time in a laser-lightintensity value. An elapsed time is obtained. A relative laser-lightintensity value of the line “control” at the obtained elapsed time isused. As a result, it is possible to control a laser-light intensityvalue in view of the phototoxicity. The same is applied to fluorescencefading.

Note that the part different from the first embodiment will only bedescribed hereinafter based on the first embodiment.

[Configuration of Microscope System]

Firstly, the configuration of the microscope system 1 a of the presenttechnology will be described schematically. Note that the microscopesystem 1 a is a laser scanning microscope system. FIG. 9 is a diagramschematically showing the configuration of the microscope system 1 a.

The third embodiment is different from the first embodiment in that thestorage 52 further stores elapsed time/laser-light intensity valuetables for phototoxicity 52 d (fourth relation information) and elapsedtime/laser-light intensity value tables for fading 52 e (fifth relationinformation).

[Five Kinds of Tables]

The storage 52 stores five kinds of tables, and the tables are used tocontrol laser light. Here, the five kinds of tables will be described.The three kinds of tables out of the five kinds of tables are same asthose of the first embodiment, i.e., the brightness/phototoxicity tables52 a, the brightness/fading tables 52 b, and the depth/laser lightdeterioration tables 52 c.

The additionally-stored elapsed time/laser-light intensity value tablefor phototoxicity 52 d shows the relation between the elapsed time andthe relative laser-light intensity value to reduce the phototoxicity.The line “control” of the graph of FIG. 7 shows this relation.

Moreover, the additionally-stored elapsed time/laser-light intensityvalue table for fading 52 e shows the relation between the elapsed timeand the relative laser-light intensity value to reduce fading. Therelation is not shown in the drawings, but is similar to the line“control” of the graph of FIG. 7.

Those tables are previously created based on a plurality ofmeasurements. In the previous measurement, the line “control” of thegraph shows the control resultant values. However, in the mainimage-taking process, the line “control” of the graph shows controltarget values.

[Processing Flow]

The processing flow of this embodiment is the same as the processingflow of the first embodiment except for obtaining an elapsed time when asample is observed and except for calculating a laser-light intensityvalue to be reset at the time of taking a fluorescence image. Because ofthis, here, the elapsed-time obtaining process and a method ofcalculating a laser-light intensity value will only be described.

(Elapsed-Time Obtaining Process)

FIG. 10 is a flowchart illustrating the entire processing flow of thecontroller 51 of the system controller PC 50. The entire processing flowof the third embodiment is different from that of the first embodimentin that an elapsed time of observation is obtained (Step S91) after alaser-light deterioration parameter is obtained in Step S90, and in thatthe process of calculating a laser-light intensity value (Step S100 a)is different from the process of Step S100.

(Method of Calculating Laser-Light Intensity Value)

Here, as described above, a laser-light intensity value is calculated inStep S100 a. A method of calculating a laser-light intensity value willbe described. FIG. 11 is a flowchart illustrating the method ofcalculating a laser-light intensity value.

First, the controller 51 obtains a first laser-light intensity valuebased on the phototoxicity parameter calculated in Step S70 (Step S101).

Next, the controller 51 obtains a second laser-light intensity valuebased on the fading parameter calculated in Step S80 (Step S102).

Next, the controller 51 determines if the first laser-light intensityvalue is larger than the second laser-light intensity value (Step S103).

If the first laser-light intensity value is larger than the secondlaser-light intensity value (Step S103, Y), the controller 51 selects afading parameter as a criterion for calculation. Then, the controller 51calculates a third laser-light intensity value based on the selectedfading parameter and the laser-light deterioration parameter (Step S104a).

Next, the controller 51 obtains a fifth laser-light intensity value inrelation to the obtained elapsed time based on the elapsedtime/laser-light intensity value table for fading 52 e (Step S106).

Next, the controller 51 compares the calculated third laser-lightintensity value with the calculated fifth laser-light intensity value.The controller 51 selects the smaller laser-light intensity value as afinal laser-light intensity value to be reset (Step S108).

To the contrary, if the first laser-light intensity value is not largerthan the second laser-light intensity value (Step S103, N), thecontroller 51 selects a phototoxicity parameter as a criterion forcalculation. Then, the controller 51 calculates a fourth laser-lightintensity value based on the selected phototoxicity parameter and thelaser-light deterioration parameter (Step S105 a).

Next, the controller 51 obtains a sixth laser-light intensity value inrelation to the obtained elapsed time based on the elapsedtime/laser-light intensity value table for phototoxicity 52 d (StepS107).

Next, the controller 51 compares the calculated fourth laser-lightintensity value with the calculated sixth laser-light intensity value.The controller 51 selects the lower laser-light intensity value as afinal laser-light intensity value to be reset (Step S109).

The method of calculating a laser-light intensity value of Step S100 ahas been described.

Modification Example 1

In the third embodiment, both brightness values and elapsed time are fedback to reset a laser-light intensity value. To the contrary, in themodification example, a laser-light intensity value is reset only basedon elapsed time.

In this case, only two kinds of tables, i.e., the elapsedtime/laser-light intensity value tables for phototoxicity 52 d and theelapsed time/laser-light intensity value tables for fading 52 e, areused to obtain a laser-light intensity value.

Note that the configuration and the processing flow of this modificationexample correspond to part of the above-mentioned configuration andprocessing flow, and detailed description thereof will be omitted.

[Notes]

Moreover, the present technology is not limited to the above-mentionedembodiments, but may be variously modified within the gist of thepresent technology, as a matter of course.

[Other Configurations of the Present Technology]

Note that the present technology may employ the followingconfigurations.

(1) A laser scanning microscope system, comprising:

a laser light source capable of emitting laser light, the laser lightbeing excitation light, a fluorescent-dyed biological sample beingirradiated with the excitation light and emitting light;

a photodetector configured

-   -   to detect fluorescence emitted by the biological sample, and    -   to output a signal corresponding to a brightness value; and

a controller apparatus including

-   -   storage configured to prestore first relation information, the        first relation information including        -   the plurality of laser-light intensity values, and        -   information on at least one possible correlation between a            phototoxicity degree and the brightness value in relation to            each of the laser-light intensity values, the phototoxicity            to the biological sample resulting from the laser light, and    -   a controller configured        -   to generate a fluorescence image having the brightness value            based on the signal output from the photodetector,        -   to calculate a brightness value representative of a ROI area            based on the generated fluorescence image, and        -   to refer to the first relation information, and to determine            a laser-light intensity value satisfying tolerance of the            phototoxicity based on the calculated representative            brightness value.            (2) The laser scanning microscope system according to (1),            wherein

the storage is configured to further prestore second relationinformation, the second relation information including

-   -   the plurality of laser-light intensity values, and    -   information on at least one possible correlation between a        fading degree of the fluorescence and the brightness value in        relation to each of the laser-light intensity values, the fading        of the fluorescence of the biological sample resulting from the        laser light, and

the controller is configured to select, based on the calculatedrepresentative brightness value, one of a laser-light intensity valuesatisfying a tolerance of the phototoxicity determined with reference tothe first relation information and a laser-light intensity valuesatisfying a tolerance of the fading determined with reference to thesecond relation information, the selected laser-light intensity valuebeing smaller than the other laser-light intensity value.

(3) The laser scanning microscope system according to (1) or (2),wherein

the storage is configured to prestore third relation information, thethird relation information showing a relation between a depth and anattenuation degree of the excitation light, the depth being between asurface of the biological sample and a position irradiated with theexcitation light, and

the controller is configured

-   -   to refer to the third relation information, and    -   to correct the selected laser-light intensity value.        (4) The laser scanning microscope system according to any one        of (1) to (3), wherein

the controller is configured to select sensitivity of the photodetectorbased on the corrected laser-light intensity value.

(5) The laser scanning microscope system according to any one of (1) to(4), wherein

the storage is configured to prestore fourth relation information, thefourth relation information showing relation between an elapsed time ofobservation of the biological sample and a laser-light intensity valuesatisfying a tolerance of phototoxicity, the phototoxicity to thebiological sample resulting from the laser light, and

the controller is configured to select one of the corrected laser-lightintensity value and a laser-light intensity value determined based onthe elapsed time of observation with reference to the fourth relationinformation, the selected laser-light intensity value being lower thanthe other laser-light intensity value.

(6) The laser scanning microscope system according to any one of (1) to(4), wherein

the storage is configured to prestore fifth relation information, thefifth relation information showing relation between an elapsed time ofobservation of the biological sample and a laser-light intensity valuesatisfying a tolerance of fading, the fading being given to thebiological sample by the laser light, and

the controller is configured to select one of the corrected laser-lightintensity value and a laser-light intensity value determined based onthe elapsed time of observation with reference to the fifth relationinformation, the selected laser-light intensity value being lower thanthe other laser-light intensity value.

(7) A method of setting a laser-light intensity value, comprising:

emitting laser light, the laser light being excitation light, afluorescent-dyed biological sample being irradiated with the excitationlight and emitting light;

detecting fluorescence emitted by the biological sample, and outputtinga signal corresponding to a brightness value;

prestoring relation information, the relation information including theplurality of laser-light intensity values, and information on at leastone possible correlation between a phototoxicity degree and thebrightness value in relation to each of the laser-light intensityvalues, the phototoxicity to the biological sample resulting from thelaser light;

generating a fluorescence image having the brightness value based on theoutput signal;

calculating a brightness value representative of a ROI area based on thegenerated fluorescence image; and

referring to the relation information, and determining a laser-lightintensity value satisfying tolerance of the phototoxicity based on thecalculated representative brightness value.

It should be understood by those skilled in the art that variousmodifications, combinations, sub-combinations and alterations may occurdepending on design requirements and other factors insofar as they arewithin the scope of the appended claims or the equivalents thereof.

What is claimed is:
 1. A microscope system, comprising: a light sourceconfigured to emit an excitation light to irradiate a fluorescent-dyedbiological sample; a photodetector configured to detect fluorescenceemitted by the biological sample, and to output a signal correspondingto the detected fluorescence; and circuitry configured to: generate afluorescence image based on the signal output from the photodetector,calculate a brightness value representative of at least a portion of thegenerated fluorescence image, access first relation information on atleast one possible correlation between a phototoxicity degree and thebrightness value in relation to each of a plurality light intensityvalues, wherein the first relation information is further related to anelapsed time of observation, determine a light intensity valuesatisfying a tolerance of the phototoxicity based on (i) the calculatedrepresentative brightness value, (ii) the first relation information,and (iii) the elapsed time of observation, and use the determined lightintensity value to control an intensity of the excitation light emittedby the light source.
 2. The microscope system according to claim 1, thesystem further comprising: a stage on which the fluorescent-dyedbiological sampled can be placed; and a stage driver configured to movethe stage three dimensionally in relation to the light source or thephotodetector.
 3. The microscope system according to claim 2, whereinthe circuitry is further configured to adjust a focus onto thebiological sample.
 4. The microscope system according to claim 2,wherein the stage driver is further configured to control movement ofthe stage based on a movement of the biological sample.
 5. Themicroscope system according to claim 4, wherein the circuitry is furtherconfigured to adjust an exposure value.
 6. The microscope systemaccording to claim 1, further comprising: a memory that stores the firstrelation information, wherein the circuitry is further configured toaccess the memory to access the first relation information.
 7. Themicroscope system according to claim 1, wherein the circuitry is furtherconfigured to determine a kind of fluorescent-dye in the biologicalsample, and to selectively access first relation information thatcorresponds to the determined kind of fluorescent-dye.
 8. The microscopesystem according to claim 7, wherein the circuitry is further configuredto determine a type of the biological sample, and to selectively accessfirst relation information that corresponds to the determined type ofthe biological sample.
 9. The microscope system according to claim 1,wherein the light source comprises a laser scanning type light sourcethat is capable of being configured to emit a wavelength of the laserlight that corresponds to the fluorescent dye.
 10. The microscope systemaccording to claim 1, wherein the first relation information is furtherrelated to a total time of excitation, and the circuitry is furtherconfigured to determine the light intensity value satisfying thetolerance of the phototoxicity based on the total time of excitation.11. The microscope system according to claim 1, wherein the circuitry isfurther configured to: receive information on at least one of (i) a typeof the biological sample, (ii) a kind of fluorescent-dye in thebiological sample, or (iii) a purpose of the image-taking, andselectively access first relation information that corresponds to thereceived information.
 12. The microscope system according to claim 1,wherein the circuitry is further configured to: set region of interestin the generated fluorescent image, and calculate the brightness valuebased on only image signals corresponding to the region of interest. 13.The microscope system according to claim 12, wherein the circuitry isfurther configured to set a block having a larger brightness densitythan other blocks as the region of interest.
 14. A method for operatinga microscope system, comprising: irradiating a fluorescent-dyedbiological sample with an excitation light; detecting fluorescenceemitted by the biological sample, and outputting a signal correspondingto the detected fluorescence; generating a fluorescence image based onthe signal output from the photodetector; calculating a brightness valuerepresentative of at least a portion of the generated fluorescenceimage; accessing first relation information on at least one possiblecorrelation between a phototoxicity degree and the brightness value inrelation to each of a plurality light intensity values, wherein thefirst relation information is further related to an elapsed time ofobservation; determining a light intensity value satisfying a toleranceof the phototoxicity based on (i) the calculated representativebrightness value, (ii) the first relation information, and (iii) theelapsed time of observation; and using the determined light intensityvalue to control an intensity of the excitation light.
 15. A microscopesystem, comprising: a light source configured to emit an excitationlight to irradiate a fluorescent-dyed biological sample; a photodetectorconfigured to detect fluorescence emitted by the biological sample, andto output a signal corresponding to the detected fluorescence; a stageon which the fluorescent-dyed biological sampled can be placed; a stagedriver configured to move the stage three dimensionally in relation tothe light source or the photodetector; and circuitry configured to:generate a fluorescence image based on the signal output from thephotodetector, calculate a brightness value representative of at least aportion of the generated fluorescence image, access information on atleast one of (i) a type of the biological sample, or (ii) a kind of thefluorescent dye in the biological sample, selectively access firstrelation information between the brightness value and light intensityvalues from a database based on the at least one of the type of thebiological sample or the kind of the fluorescent dye in the biologicalsample, wherein the first relation information is further related to anelapsed time of observation, determine a light intensity value based on(i) the calculated representative brightness value, (ii) the firstrelation information, and (iii) the elapsed time of observation, and usethe determined light intensity value to control an intensity of theexcitation light emitted by the light source.